Cell migration is central to numerous biological and pathological processes, such as cancer. The migration of cancer cells away from the primary tumor into surrounding tissue is an important initial step in tumor progression. Contributions from the tumor microenvironment, including cell types other than the cancer cells and the organization of the surrounding extracellular matrix (ECM), are increasingly recognized in regulating the migratory/invasive properties of cancer cells. Cancer-associated fibroblasts (CAFs) are emerging as an important cell type in the tumor microenvironment because of their role in regulating tumor-stroma interactions and tumor progression. However, the role of CAFs in directing cancer cell migration and the underlying mechanisms that regulate this process are not well understood. The endosomal adaptor protein APPL1 has been implicated in cancer; however, its role in cancer cell migration is currently poorly understood. My preliminary data points to a new role for APPL1 in regulating cancer cell migration. The goal of this proposal is to elucidate the mechanism whereby APPL1 regulates cancer cell migration and the contribution of the tumor microenvironment to this process. We have preliminary data demonstrating that APPL1 decreases Rac activation, and so Aim I will probe the mechanism by which APPL1 regulates Rac activation. The expression of APPL1 will be altered in HT1080 fibrosarcoma cells and the relative levels of active Rac GTPase will be assessed. APPL1 binds Rab5, a critical GTPase for the regulation of vesicular trafficking of receptors, including integrins. Thus, APPL1 could also modulate integrin trafficking through its interaction with Rab5 and thereby regulate cancer cell migration. In Aim II, studies will be performed to investigate the role of APPL1 in integrin trafficking. Live-cell microscopy will be used to observe integrin trafficking in cells eiher expressing APPL1-GFP, or shRNAs targeted against APPL1, using various a5 integrin constructs. Integrin internalization and recycling assays will be used to quantify integrin trafficking in cells expressing APPL1-GFP as compared to GFP control or APPL1 knockdown. Alterations of the tumor microenvironment are being increasingly studied in the role of regulating cancer cell migration/invasion, which can then lead to cancer cell metastasis. Furthermore, we have preliminary data indicating that CAFs alter the alignment of the ECM protein, fibronectin (FN), through Rac activation. It is possible that APPL1 regulates these processes. Therefore, Aim III will determine whether APPL1 regulates CAF-mediated alterations of the tumor microenvironment and directed cell migration. I will co-culture SCC61 head and neck squamous carcinoma cells with human primary CAFs expressing APPL1-GFP or normal associated fibroblasts (NAFs) expressing shRNAs targeted against APPL1, and assess various parameters of migration. The proposed studies will provide important insight into the molecular mechanisms that regulate the tumor microenvironment and cancer cell migration, which could lead to the discovery of new therapeutic options for cancer.